ENGINEERING PROKARYOTIC PHOSHOLIPASE A2

Abstract

PROKARYOTIC PLA2 FROM STREPTOMYCES VIOLACEORUBER, WHEN OVEREXPRESSED IN E. COLI USING THE PELB PERIPLASMIC SIGNAL, WAS FOUND TO BE SECRETED INTO THE CULTURE MEDIUM, INSTEAD OF ITS RETENTION IN THE BACTERIAL PERIPLASMIC SPACE. THIS MOLECULE IS STRUCTURALLY THE LEAST COMPLEX AMONG PLA2 ENZYMES. HENCE, IN MY DOCTORAL STUDY I HAVE ATTEMPTED TO ENGINEER PLA2 TO (A) DEVELOP AN EXPRESSION SYSTEM; AND (B) DESIGN A SMALLER SCAFFOLD. THE SECRETORY NATURE OF PROKARYOTIC PLA2 WAS PUT TO USE BY INTRODUCING IT AS A TAG TO EXPRESS DISULPHIDE RICH PROTEINS AS IT WOULD PROVIDE AN OXIDATIVE ENVIRONMENT FOR FOLDING. IN THIS SYSTEM, WE SUCCESSFULLY EXPRESSED FUSION PROTEIN PLA2-KRAIT NATRIURETIC PEPTIDE (ONE DISULPHIDE) AND PLA2-DENMOTOXIN (A THREE-FINGER TOXIN WITH FIVE DISULPHIDES) IN THE CULTURE MEDIUM. AFTER CLEAVAGE, CORRECTLY FOLDED, FUNCTIONALLY-ACTIVE KRAIT NATRIURETIC PEPTIDE WAS OBTAINED. THUS, WE DEMONSTRATED THE UTILITY OF PROKARYOTIC PLA2 IN THE EXPRESSION OF COMPLEX PROTEINS. THIS PLA2 H